Animals have diversified and adapted to fill the numerous habitats and environments on this earth. The long-term goal of our studies is to elucidate mechanisms that drive these adaptive changes at the molecular and functional levels. We plan to accomplish this goal using vision as a model system. To identify critical amino acid (AA) changes that modify the wavelengths of maximal absorption (;maxs) of visual pigments, vision scientists analyze contemporary pigments. Experimental evolutionary biologists also manipulate contemporary pigments and infer the past. However, by ignoring the evolutionary processes of visual pigments, neither the molecular basis of spectral tuning in visual pigments nor the evolutionary mechanisms of visual pigments can be elucidated. Fortunately, the central unanswered questions in phototransduction and evolutionary biology can be solved simultaneously by genetically engineering and manipulating proper ancestral pigments. Here, we propose to elucidate the molecular mechanisms that drive adaptive evolution of RH2, SWS1, and SWS2 pigments in vertebrates, which have ;maxs of ~450-530, ~355-440, and ~400-460 nm, respectively. We approach the problem not only by determining the molecular basis of spectral tuning in visual pigments but also by establishing the relationships between organisms with different sets of visual pigments and their ecological environments. For each pigment group, we plan to 1) infer the AA sequences of ~15 ancestral pigments and engineer them and determine their ;maxs, 2) infer AA changes that shift the ;max, 3) identify the critical AA changes by mutating ancestral pigments, and 4) establish the universal chemical principle of the spectral tuning in visual pigments by using the quantum mechanical/molecular mechanical (QM/MM) methods, which have been proven to be highly effective. We shall also test the possibility of adaptive evolution of these visual pigments by examining whether the directions of the ;max-shifts of visual pigments in various species match with the changes in the organisms' ecological environments. For this purpose, each of the three pigment groups with variable ;maxs in multiple species will be classified into distinct classes according to their environments and tested for the possibility of adaptive evolution. Since virtually all fish SWS1 pigments examined to date are UV-sensitive, we also plan to search for violet-sensitive SWS1 pigments in different fishes, by surveying fishes that live at depths of 0-200 m and prey on small fishes and do not require UV vision for hunting. Considering the strong AA interactions that occur in SWS1 pigments, we also plan to elucidate how various critical AA changes could have accumulated during its evolution and learn the chemical structural preconditions required for UV pigments to become violet-sensitive and vice versa.